Production of pulse protein product with reduced astringency

ABSTRACT

Pulse proteins of reduced astringency are obtained by fractionating pulse protein products which are completely soluble and heat stable in aqueous media at acid pH value of less than about 4.4 into lower molecular weight, less astringent proteins and higher molecular weight, more astringent proteins.

REFERENCE TO RELATED APPLICATIONS

This application is a division of copending U.S. patent application Ser.No. 14/290,415 filed May 29, 2014 which itself claims priority under 35USC 119 (e) from U.S. Provisional Patent Application No. 61/828,735filed May 30, 2013 and 61/927,182 filed Jan. 14, 2014.

FIELD OF THE INVENTION

The present invention relates to the production of pulse proteinproducts, preferably pulse protein isolates.

BACKGROUND TO THE INVENTION

In U.S. patent application Ser. No. 13/103,528 filed May 9, 2011 (USPatent Publication No. 2011-027497 published Nov. 10, 2011), Ser. No.13/289,264 filed Nov. 4, 2011 (US Patent Publication No. 2012-0135117published May 31, 2012), Ser. No. 13/556,357 filed Jul. 24, 2012 (USPatent Publication No. 2013-0189408 published Jul. 25, 2013) and Ser.No. 13/642,003 filed Jan. 7, 2013 (US Patent Publication No.2013-0129901 published May 23, 2013), assigned to the assignee hereofand the disclosures of which are incorporated herein by reference, thereis described the provision of a novel pulse protein product having aprotein content of at least about 60 wt % (N×6.25) on a dry weightbasis, preferably a pulse protein isolate having a protein content of atleast about 90 wt % (N×6.25) d.b. The pulse protein product has a uniquecombination of properties, namely:

-   -   completely soluble in aqueous media at acid pH values of less        than about 4.4    -   heat stable in aqueous media at acid pH values of less than        about 4.4    -   does not require stabilizers or other additives to maintain the        protein product in solution    -   is low in phytic acid    -   requires no enzymes in the production thereof

This novel pulse protein product is prepared by a method whichcomprises:

-   -   extracting a pulse protein source with an aqueous calcium salt        solution, preferably an aqueous calcium chloride solution, to        cause solubilization of pulse protein from the protein source        and to form an aqueous pulse protein solution,    -   (b) separating the aqueous pulse protein solution from residual        pulse protein source,    -   (c) optionally diluting the aqueous pulse protein solution,    -   (d) adjusting the pH of the aqueous pulse protein solution to a        pH of about 1.5 to about 4.4, preferably about 2 to about 4, to        produce an acidified pulse protein solution,    -   (e) optionally clarifying the acidified pulse protein solution        if it is not already clear,    -   (f) alternatively from steps (b) to (e), optionally, diluting        and then adjusting the pH of the combined aqueous pulse protein        solution and residual pulse protein source to a pH of about 1.5        to about 4.4, preferably about 2 to about 4, then separating the        acidified, preferably clear, pulse protein solution from        residual pulse protein source,    -   (g) optionally concentrating the aqueous pulse protein solution        while maintaining the ionic strength substantially constant by a        selective membrane technique,    -   (h) optionally diafiltering the optionally concentrated pulse        protein solution, and    -   (i) optionally drying the optionally concentrated and optionally        diafiltered pulse protein solution.

The pulse protein product preferably is an isolate having a proteincontent of at least about 90 wt %, preferably at least about 100 wt %(N×6.25) d.b.

In certain acidic beverages, particularly those having a pH at the lowend of the acceptable pH range for acidic beverages, the novel pulseprotein product tends to induce an undesirable astringent sensation inthe mouth.

SUMMARY OF THE INVENTION

It has now been found that this undesirable astringency can be reducedor eliminated by modifying the procedure used to manufacture the novelpulse protein product.

In accordance with the present invention, there is provided a method ofpreparing pulse protein product with reduced astringency, whichcomprises:

-   -   (a) extracting a pulse protein source with an aqueous calcium        salt solution to cause solubilization of pulse protein from the        protein source and to form an aqueous pulse protein solution,    -   (b) separating the aqueous pulse protein solution from residual        pulse protein source,    -   (c) optionally diluting the aqueous pulse protein solution,    -   (d) adjusting the pH of the aqueous pulse protein solution to a        pH of about 1.5 to about 4.4 to produce an acidified pulse        protein solution,    -   (e) optionally clarifying the acidified pulse protein solution        if it is not already clear,    -   (f) alternatively from steps (b) to (e), optionally, diluting        and then adjusting the pH of the combined aqueous pulse protein        solution and residual pulse protein source to a pH of about 1.5        to about 4.4 and then separating the acidified, preferably        clear, pulse protein solution from residual pulse protein        source, and    -   (g) fractionating the proteins in the acidified pulse protein        solution to separate lower molecular weight, less astringent        proteins from higher molecular weight, more astringent proteins.

In accordance with one aspect of the present invention, the process ismodified to remove proteins which precipitate at a pH of about 5 toabout 6.5 and that may interact with salivary proteins, therebyproducing a less astringent product. In order to precipitate the proteinfraction, the pH of the acidified pulse protein solution, preferablyafter partial concentration and diafiltration, is adjusted to about 5 toabout 6.5, preferably about 5.5 to about 6.0. The precipitated proteinis removed and the protein that remains in solution is then re-acidifiedto about pH 3 and further membrane processed to form one of the productsof the invention. The collected material that precipitates upon pHadjustment may be further processed to provide another product of theinvention The precipitated material may be processed as follows:

-   -   1. Optionally washed with water and spray dried at about pH 5.5        or    -   2. Optionally washed with water, adjusted to a pH of about 6 to        8 then spray dried, or    -   3. Adjusted to about pH 3, membrane processed then spray dried,        or    -   4. Adjusted to about pH 3, membrane processed, adjusted in pH to        about 6 to 8 then spray dried

This product is intended typically for use in neutral applications.

The less astringent proteins that remain in solution when theaforementioned precipitation method is applied seem to be of lowermolecular weight than the more astringent species. In another aspect ofthe present invention, the more astringent protein component may beseparated from the less astringent protein component by membraneprocessing. Concentration and optional diafiltration of a proteinsolution containing a mixture of the more and less astringent proteinsusing a membrane with an appropriate pore size allows the smaller, lessastringent proteins to pass through with the permeate, while retainingthe more astringent species in the concentrated protein solution. Theless astringent proteins may be separated from the contaminants by asubsequent ultrafiltration and/or diafiltration step using a membranehaving a smaller pore size than that employed in the fractionation step.The purified less astringent protein fraction is a product of theinvention. The solution of larger, more astringent protein species mayalso be further processed and optionally neutralized to form anotherproduct of the invention, which is typically intended for use in neutralapplications.

In accordance with a further aspect of the present invention, there isprovided a pulse protein product having a protein content of at leastabout 60% wt % (N×6.25) d.b. and which

-   -   is completely soluble in aqueous media at acid pH values of less        than 4.4    -   is heat stable in aqueous media at acid values of less than        about 4.4    -   does not require stabilizers or other additives to maintain the        protein product in solution or suspension    -   is low in phytic acid    -   requires no enzymes in the production thereof    -   is low in astringency when tasted in aqueous solution at a pH        below about 5.

The pulse protein product preferably has a protein content of at leastabout 90 wt %, more preferably 100 wt %, (N×6.25) d.b. The pulse proteinproduct preferably is not hydrolysed and preferably has a phytic acidcontent of less than about 1.5 wt %, preferably less than about 0.5 wt%.

In accordance with a further aspect of the present invention, there isprovided a pulse protein product having a protein content of at leastabout 60 wt % (N×6.25) d.b. and having low astringency when tasted inaqueous solution at a pH of below about 5 which is substantiallycompletely soluble in an aqueous medium at a pH of less than about 4.4.

The pulse protein product may be blended with water-soluble powderedmaterials for the production of aqueous solutions of the blend,preferably a powdered beverage. The pulse protein product may be formedwith an aqueous solution, such as, a beverage, which is heat stable at atemperature of less than about 4.4. The beverage may be a clear beveragein which the dissolved pulse protein product is completely soluble andtransparent or may be a non-transparent beverage in which the dissolvedpulse protein does or does not increase the cloud or haze level.

In accordance with a further aspect of the present invention, there isprovided a pulse protein product having a molecular weight profile, asdetermined by the methods described in Example 25, which is

about 10 to about 75% greater than about 100,000 Da

about 10 to about 45% from about 15,000 to about 100,000 Da

about 8 to about 55% from about 5,000 to about 15,000 Da

about 2 to about 12% from about 1,000 to about 5,000 Da.

The molecular weight profile may be:

about 15 to about 40% greater than about 100,000 Da

about 25 to about 40% from about 15,000 to about 100,000 Da

about 15 to about 50% from about 5,000 to about 15,000 Da

about 3 to about 10% from about 1,000 to about 5,000 Da.

In accordance with another aspect of the present invention, there isprovided a pulse protein product having a molecular weight profile, asdetermined by the methods described in Example 25, which is

about 10 to about 85% greater than about 100,000 Da

about 10 to about 45% from about 15,000 to about 100,000 Da

about 0 to about 40% from about 5,000 to about 15,000 Da

about 1 to about 34% from about 1,000 to about 5,000 Da.

The molecular weight profile may be:

about 18 to about 78% greater than about 100,000 Da

about 15 to about 38% from about 15,000 to about 100,000 Da

about 2 to about 35% from about 5,000 to about 15,000 Da

about 3 to about 25% from about 1,000 to about 5,000 Da.

In accordance with a yet further aspect of the present invention, thereis provided a pulse protein product which has a protein content of atleast about 60 wt % (N×6.25) d.b. which has a solubility at 1% proteinw/v in water at a pH of about 2 to about 7 of greater than about 50%, asdetermined by the methods described in Example 5. The pulse proteinproduct preferably has a protein content of at least about 90 wt %, morepreferably at least about 100 wt % (N×6.25) d.b.

The less astringent pulse protein products of the invention, producedaccording to the processes herein are suitable, not only for proteinfortification of acid media, but may be used in a wide variety ofconventional applications of protein products, including but not limitedto protein fortification of processed foods and beverages,emulsification of oils and as a foaming agent in products which entrapgases. The pulse protein products may also be used in nutritionalsupplements. The pulse protein products may also be used in dairyanalogue or dairy alternative products or products that are dairy/plantingredient blends. Other uses of the pulse protein products are in petfoods, animal feed and in industrial and cosmetic applications and inpersonal care products.

GENERAL DESCRIPTION OF INVENTION

The initial step of the process of providing the pulse protein productsinvolves solubilizing pulse protein from a pulse protein source. Thepulses to which the invention may be applied include, but are notlimited to lentils, chickpeas, dry peas and dry beans. The pulse proteinsource may be pulses or any pulse product or by-product derived from theprocessing of pulses. For example, the pulse protein source may be aflour prepared by grinding an optionally dehulled pulse. As anotherexample, the pulse protein source may be a protein-rich pulse fractionformed by dehulling and grinding a pulse and then air classifying thedehulled and ground material into starch-rich and protein-richfractions. The pulse protein product recovered from the pulse proteinsource may be the protein naturally occurring in pulses or theproteinaceous material may be a protein modified by genetic manipulationbut possessing characteristic hydrophobic and polar properties of thenatural protein.

Protein solubilization from the pulse protein source material iseffected most conveniently using calcium chloride solution, althoughsolutions of other calcium salts may be used. In addition, otheralkaline earth metal compounds may be used, such as magnesium salts.Further, extraction of the pulse protein from the pulse protein sourcemay be effected using calcium salt solution in combination with anothersalt solution, such as sodium chloride. Additionally, extraction of thepulse protein from the pulse protein source may be effected using wateror other salt solution, such as sodium chloride, with calcium saltsubsequently being added to the aqueous pulse protein solution producedin the extraction step. Precipitate formed upon addition of the calciumsalt is removed prior to subsequent processing.

As the concentration of the calcium salt solution increases, the degreeof solubilization of protein from the pulse protein source initiallyincreases until a maximum value is achieved. Any subsequent increase insalt concentration does not increase the total protein solubilized. Theconcentration of calcium salt solution which causes maximum proteinsolubilization varies depending on the salt concerned. It is usuallypreferred to utilize a concentration value less than about 1.0 M, andmore preferably a value of about 0.10 to about 0.15 M.

In a batch process, the salt solubilization of the protein is effectedat a temperature of from about 1° to about 100° C., preferably about 15°C. to about 65° C., more preferably about 20° to about 35° C.,preferably accompanied by agitation to decrease the solubilization time,which is usually about 1 to about 60 minutes. It is preferred to effectthe solubilization to extract substantially as much protein from thepulse protein source as is practicable, so as to provide an overall highproduct yield.

In a continuous process, the extraction of the protein from the pulseprotein source is carried out in any manner consistent with effecting acontinuous extraction of protein from the pulse protein source. In oneembodiment, the pulse protein source is continuously mixed with thecalcium salt solution and the mixture is conveyed through a pipe orconduit having a length and at a flow rate for a residence timesufficient to effect the desired extraction in accordance with theparameters described herein. In such a continuous procedure, the saltsolubilization step is effected in a time of about 1 minute to about 60minutes, preferably to effect solubilization to extract substantially asmuch protein from the pulse protein source as is practicable. Thesolubilization in the continuous procedure is effected at temperaturesbetween about 1° and about 100° C., preferably between about 15° C. andabout 65° C., more preferably between about 20° and about 35° C.

The extraction is generally conducted at a pH of about 4.5 to about 11,preferably about 5 to about 7. The pH of the extraction system (pulseprotein source and calcium salt solution) may be adjusted to any desiredvalue within the range of about 4.5 to about 11 for use in theextraction step by the use of any convenient food grade acid, usuallyhydrochloric acid or phosphoric acid, or food grade alkali, usuallysodium hydroxide, as required.

The concentration of pulse protein source in the calcium salt solutionduring the solubilization step may vary widely. Typical concentrationvalues are about 5 to about 15% w/v.

The protein extraction step with the aqueous salt solution has theadditional effect of solubilizing fats which may be present in the pulseprotein source, which then results in the fats being present in theaqueous phase.

The protein solution resulting from the extraction step generally has aprotein concentration of about 5 to about 50 g/L, preferably about 10 toabout 50 g/L.

The aqueous calcium salt solution may contain an antioxidant. Theantioxidant may be any convenient antioxidant, such as sodium sulfite orascorbic acid. The quantity of antioxidant employed may vary from about0.01 to about 1 wt % of the solution, preferably about 0.05 wt %. Theantioxidant serves to inhibit oxidation of any phenolics in the proteinsolution.

The aqueous phase resulting from the extraction step then may beseparated from the residual pulse protein source, in any convenientmanner, such as by employing a decanter centrifuge, followed by disccentrifugation and/or filtration, to remove residual pulse proteinsource material. The separation step may be conducted at any temperaturewithin the range of about 1° to about 100° C., preferably about 15° toabout 65° C., more preferably about 20° to about 35° C. Alternatively,the optional dilution and acidification steps described below may beapplied to the mixture of aqueous pulse protein solution and residualpulse protein source, with subsequent removal of the residual pulseprotein source material by the separation step described above. Theseparated residual pulse protein source may be dried for disposal orfurther processed, such as to recover starch and/or residual protein.Residual protein may be recovered by re-extracting the separatedresidual pulse protein source with fresh calcium salt solution and theprotein solution yielded upon clarification combined with the initialprotein solution for further processing as described below.Alternatively, the separated residual pulse protein source may beprocessed by a conventional isoelectric precipitation process or anyother convenient procedure to recover residual protein.

The aqueous pulse protein solution may be treated with an anti-foamer,such as any suitable food-grade, non-silicone based anti-foamer, toreduce the volume of foam formed upon further processing. The quantityof anti-foamer employed is generally greater than about 0.0003% w/v.Alternatively, the anti-foamer in the quantity described may be added inthe extraction steps.

The separated aqueous pulse protein solution may be subject to adefatting operation, if required, as described in U.S. Pat. Nos.5,844,086 and 6,005,076, assigned to the assignee hereof and thedisclosures of which are incorporated herein by reference.Alternatively, defatting of the separated aqueous pulse protein solutionmay be achieved by any other convenient procedure.

The aqueous pulse protein solution may be treated with an adsorbent,such as powdered activated carbon or granulated activated carbon, toremove colour and/or odour compounds. Such adsorbent treatment may becarried out under any convenient conditions, generally at the ambienttemperature of the separated aqueous protein solution. For powderedactivated carbon, an amount of about 0.025% to about 5% w/v, preferablyabout 0.05% to about 2% w/v, is employed. The adsorbing agent may beremoved from the pulse protein solution by any convenient means, such asby filtration.

The resulting aqueous pulse protein solution may be diluted generallywith about 0.1 to about 10 volumes, preferably about 0.5 to about 2volumes of aqueous diluent, in order to decrease the conductivity of theaqueous pulse protein solution to a value of generally below about 105mS, preferably about 4 to about 21 mS. Such dilution is usually effectedusing water, although dilute salt solution, such as sodium chloride orcalcium chloride, having a conductivity up to about 3 mS, may be used.

The diluent with which the pulse protein solution is mixed generally hasthe same temperature as the pulse protein solution, but the diluent mayhave a temperature of about 1° to about 100° C., preferably about 15° toabout 65° C., more preferably about 20° to about 35° C.

The optionally diluted pulse protein solution then is adjusted in pH toa value of about 1.5 to about 4.4, preferably about 2 to about 4, by theaddition of any suitable food grade acid, such as hydrochloric acid orphosphoric acid, to result in an acidified aqueous pulse proteinsolution, preferably a clear acidified aqueous pulse protein solution.The acidified aqueous pulse protein solution has a conductivity ofgenerally below about 110 mS for a diluted pulse protein solution, orgenerally below about 115 mS for an undiluted pulse protein solution, inboth cases preferably about 4 to about 26 mS.

As mentioned above, as an alternative to the earlier separation of theresidual pulse protein source, the aqueous pulse protein solution andthe residual pulse protein source material, may be optionally dilutedand acidified together and then the acidified aqueous pulse proteinsolution is clarified and separated from the residual pulse proteinsource material by any convenient technique as discussed above. Theacidified aqueous pulse protein solution may optionally be defatted,optionally treated with an adsorbent and optionally treated withdefoamer as described above.

The acidified aqueous pulse protein solution may be subjected to a heattreatment to inactivate heat labile anti-nutritional factors, such astrypsin inhibitors, present in such solution as a result of extractionfrom the pulse protein source material during the extraction step. Sucha heating step also provides the additional benefit of reducing themicrobial load. Generally, the protein solution is heated to atemperature of about 70° to about 160° C., preferably about 80° to about120° C., more preferably about 85° to about 95° C., for about 10 secondsto about 60 minutes, preferably about 10 seconds to about 5 minutes,more preferably about 30 seconds to about 5 minutes. The heat treatedacidified pulse protein solution then may be cooled for furtherprocessing as described below, to a temperature of about 2° to about 65°C., preferably about 50° C. to about 60° C.

If the optionally diluted, acidified and optionally heat treated pulseprotein solution is not transparent it may be clarified by anyconvenient procedure such as filtration or centrifugation.

In accordance with one aspect of the present invention, the acidifiedaqueous pulse protein solution, preferably following the concentrationand diafiltration steps described below, more preferably followingeffecting partial concentration and diafiltration steps described below,is adjusted in pH to the range of about 5 to about 6.5, preferably about5.5 to about 6.0 to effect protein precipitation and fractionation. SuchpH adjustment may be effected using any convenient food grade alkali,such as aqueous sodium hydroxide solution. The protein that precipitatesat such pH is collected by any convenient means such as centrifugationand the resulting solution is re-acidified to a pH of about 1.5 to about4.4, preferably about 2 to about 4, by the addition of any suitable foodgrade acid, such as hydrochloric acid or phosphoric acid, to result in are-acidified aqueous pulse protein solution, preferably a clearre-acidified aqueous pulse protein solution. This re-acidified aqueouspulse protein solution contains the less astringent protein species. There-acidified aqueous pulse protein solution then is processed accordingto the steps described below.

The protein precipitated at about pH 5 to about 6.5 and separated fromthe resulting solution may be further processed. The precipitate, whichis the more astringent protein fraction, may be washed with water andthen dried by any convenient procedure such as spray drying or freezedrying. Alternatively, the precipitate may be washed with water,adjusted in pH to about 6 to 8 and then dried. The precipitate may beadjusted to a pH of about 1.5 to about 4.4, preferably about 2 to about4, then membrane processed as described below and dried. The precipitatemay be adjusted to a pH of about 1.5 to about 4.4, preferably about 2 toabout 4, membrane processed as described below, adjusted in pH to about6 to about 8, and then dried.

The acidified aqueous pulse protein solution may be concentrated priorto fractionation by pH adjustment as described above. Such aconcentration step increases the protein concentration of the solutionwhile maintaining the ionic strength thereof substantially constant.Such a concentration step generally is effected to provide aconcentrated pulse protein solution having a protein concentration ofabout 50 to about 300 g/L, preferably about 100 to about 200 g/L. Whenthe acidified aqueous protein solution is partially concentrated beforeprecipitation and removal of the more astringent protein at pH about 5to about 6.5, the concentration step is effected preferably to a proteinconcentration of below about 50 g/L. The concentrated or partiallyconcentrated acidified aqueous solution may be diluted with water priorto the pH adjustment step in order to reduce the viscosity of the sampleand facilitate the recovery of the protein precipitated by the pHadjustment.

The re-acidified aqueous pulse protein solution may also be concentratedto increase the protein concentration thereof while maintaining theionic strength thereof substantially constant. Such a concentration stepgenerally is effected to provide a concentrated re-acidified pulseprotein solution having a protein concentration of about 10 to about 300g/L, preferably about 100 to about 200 g/L. When the re-acidifiedaqueous protein solution is partially concentrated, the concentrationstep is effected preferably to a protein concentration of less thanabout 10 g/L.

Such concentration steps may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 1,000 to about 1,000,000 daltons, preferably about 1,000 toabout 100,000 daltons, more preferably about 1,000 to about 10,000daltons having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass therethroughwhile preventing higher molecular weight species from so doing. The lowmolecular weight species include not only the ionic species of the saltbut also low molecular weight materials extracted from the sourcematerial, such as carbohydrates, pigments and low molecular weightproteins including the less astringent proteins (discussed below) andthe anti-nutritional trypsin inhibitors. The molecular weight cut-off ofthe membrane is usually chosen to ensure retention of a significantproportion of the protein in the solution, while permitting contaminantsto pass through having regard to the different membrane materials andconfigurations.

The concentrated acidified or concentrated re-acidified pulse proteinsolution may be subjected to a diafiltration step using water or adilute saline solution. The diafiltration solution may be at its naturalpH or at a pH equal to that of the protein solution being diafiltered orat any pH value in between. Such diafiltration may be effected usingfrom about 1 to about 40 volumes of diafiltration solution, preferablyabout 2 to about 25 volumes of diafiltration solution. In thediafiltration operation, further quantities of contaminants are removedfrom the aqueous pulse protein solution by passage through the membranewith the permeate. This purifies the aqueous protein solution and mayalso reduce its viscosity. The diafiltration operation may be effecteduntil no significant further quantities of contaminants or visiblecolour are present in the permeate or in the case of the re-acidifiedprotein solution, until the retentate has been sufficiently purified soas, when dried, to provide a pulse protein isolate with a proteincontent of at least about 90 wt % (N×6.25) d.b. Such diafiltration maybe effected using the same membrane as for the concentration step.However, if desired, the diafiltration step may be effected using aseparate membrane with a different molecular weight cut-off, such as amembrane having a molecular weight cut-off in the range of about 1,000to about 1,000,000 daltons, preferably about 1,000 to about 100,000daltons, more preferably about 1,000 to about 10,000 daltons havingregard to different membrane materials and configuration.

Alternatively, the diafiltration step may be applied to the acidified orre-acidified aqueous protein solution prior to concentration or topartially concentrated acidified or partially concentrated re-acidifiedaqueous protein solution. Diafiltration may also be applied at multiplepoints during the concentration process. When diafiltration is appliedprior to concentration or to partially concentrated solution, theresulting diafiltered solution may then be fully concentrated. Theviscosity reduction achieved by diafiltering multiple times as theprotein solution is concentrated may allow a higher final, fullyconcentrated protein concentration to be achieved. In the case of there-acidified protein solution, this reduces the volume of material to bedried.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated pulse proteinsolution.

The concentration steps and the optional diafiltration steps may beeffected at any convenient temperature, generally about 2° to about 65°C., preferably about 50° to about 60° C., and for the period of time toeffect the desired degree of concentration. The temperature and otherconditions used to some degree depend upon the membrane equipment usedto effect the membrane processing, the desired protein concentration ofthe solution and the efficiency of the removal of contaminants to thepermeate.

In accordance with another aspect of this invention, the concentrationand optional diafiltration steps are operated on the aqueous acidifiedpulse protein solution in such a way as to separate the lower molecularweight, less astringent proteins from the higher molecular weight, moreastringent proteins. When this process is employed the molecular weightcut-off of the concentration and diafiltration membranes are chosen topermit the smaller, less astringent proteins to pass to the permeatewith the contaminants. Such concentration and diafiltration steps may beeffected in any convenient manner consistent with batch or continuousoperation, such as by employing any convenient selective membranetechnique, such as microfiltration or ultrafiltration, using membranes,such as hollow-fibre membranes or spiral-wound membranes, with asuitable molecular weight cut-off, such as about 0.05 to about 0.1 μm,preferably about 0.08 to about 0.1 μm for microfiltration and about10,000 to about 1,000,000 daltons, preferably about 100,000 to about1,000,000 daltons for ultrafiltration, having regard to differingmembrane materials and configurations, and, for continuous operation,dimensioned to permit the desired degree of concentration as the aqueousprotein solution passes through the membranes. In the concentration stepthe acidified protein solution is concentrated to a proteinconcentration of about 50 to about 300 g/L, preferably about 100 toabout 200 g/L. The concentrated protein solution then may be diafilteredwith water or dilute salt solution. The diafiltration solution may be atits natural pH or at a pH equal to that of the protein solution beingdiafiltered or any pH value in between. Such diafiltration may beeffected using from about 1 to about 40 volumes of diafiltrationsolution, preferably about 2 to about 25 volumes of diafiltrationsolution. The concentration and optional diafiltration steps may beeffected at any convenient temperature, generally about 2° to about 65°C., preferably about 50° to about 60° C. The smaller less astringentproteins are captured in the permeate of the membrane processes alongwith other small molecule contaminants.

The less astringent proteins are then separated from the contaminants bysubsequent concentration of the protein solution (step 1 permeate) bymembrane processing such as ultrafiltration to a protein concentrationof about 10 to about 300 g/L, preferably about 100 to about 200 g/L andoptional diafiltration. When the protein solution (step 1 permeate) ispartially concentrated, the concentration step is effected preferably toa protein concentration of less than about 10 g/L. The concentration anddiafiltration steps are performed using a membrane having a lowermolecular weight cut-off such as about 1,000 to about 100,000 daltons,preferably 1,000 to about 10,000 dalton operated as described above.

Additional products may be obtained from the retentate of the membranefractionation process, which contains the more astringent proteins. Thisprotein solution may be dried by any convenient means, with or withoutadjustment of the pH of the protein solution to about 6 to about 8 usingfood grade alkali.

The concentration and the optional diafiltration steps employed in thepurification of the aqueous solutions of less astringent proteinsderived from either the precipitation or membrane fractionationprocedure may be effected herein in such a manner that the lessastringent pulse protein product recovered contains less than about 90wt % protein (N×6.25) d.b., such as at least about 60 wt % protein(N×6.25) d.b. By partially concentrating and/or partially diafilteringthe aqueous pulse protein solution, it is possible to only partiallyremove contaminants. This protein solution may then be dried to providea pulse protein product with lower levels of purity. The pulse proteinproduct is highly soluble and able to produce less astringent proteinsolutions, preferably clear, less astringent protein solutions, underacidic conditions.

As alluded to earlier, pulses contain anti-nutritional trypsininhibitors. The level of trypsin inhibitor activity in the final pulseprotein product can be controlled by the manipulation of various processvariables.

As noted above, heat treatment of the acidified aqueous pulse proteinsolution may be used to inactivate heat-labile trypsin inhibitors. Thepartially concentrated or fully concentrated acidified pulse proteinsolution may also be heat treated to inactivate heat labile trypsininhibitors. Such a heat treatment may also be applied to there-acidified pulse protein solution arising from the precipitationfractionation method or the solution of less astringent, lower molecularweight proteins arising from the membrane separation method, before orafter partial or complete concentration. When the heat treatment isapplied to a solution that is not already fully concentrated, theresulting heat treated solution may then be additionally concentrated.

Acidifying and membrane processing the pulse protein solution at a lowerpH, such as 1.5 to 3, may reduce the trypsin inhibitor activity relativeto processing the solution at higher pH, such as 3 to 4.4. When theprotein solution is concentrated and diafiltered at the low end of thepH range, it may be desired to raise the pH of the retentate prior todrying. The pH of the concentrated and diafiltered protein solution maybe raised to the desired value, for example pH 3, by the addition of anyconvenient food grade alkali, such as sodium hydroxide.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing pulse materials to reducing agents that disrupt or rearrangethe disulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added with the pulseprotein source material in the extraction step, may be added to theclarified aqueous pulse protein solution following removal of residualpulse protein source material, may be added to the optionallydiafiltered retentate before drying or may be dry blended with the driedpulse protein product. The addition of the reducing agent may becombined with the heat treatment step and membrane processing steps, asdescribed above.

If it is desired to retain active trypsin inhibitors in the products,this can be achieved by eliminating or reducing the intensity of theheat treatment step, not utilizing reducing agents, operating theconcentration and diafiltration steps at the higher end of the pH range,such as 3 to 4.4.

Any of the concentrated and optionally diafiltered protein solutionsdescribed above may be subject to a further defatting operation, ifrequired, as described in U.S. Pat. Nos. 5,844,086 and 6,005,076.Alternatively, defatting of the concentrated and optionally diafilteredprotein solutions may be achieved by any other convenient procedure.

Any of the concentrated and optionally diafiltered aqueous proteinsolutions described above may be treated with an adsorbent, such aspowdered activated carbon or granulated activated carbon, to removecolour and/or odour compounds. Such adsorbent treatment may be carriedout under any convenient conditions, generally at the ambienttemperature of the concentrated protein solution. For powdered activatedcarbon, an amount of about 0.025% to about 5% w/v, preferably about0.05% to about 2% w/v, is employed. The adsorbent may be removed fromthe pulse protein solution by any convenient means, such as byfiltration.

The concentrated and optionally diafiltered aqueous pulse proteinsolutions or collected pulse protein precipitates described above may bedried by any convenient technique, such as spray drying or freezedrying. A pasteurization step may be effected on the pulse proteinsolutions or resuspended pulse protein precipitates prior to drying.Such pasteurization may be effected under any desired pasteurizationconditions. Generally, the concentrated and optionally diafiltered pulseprotein solution or resuspended pulse protein precipitate is heated to atemperature of about 55° to about 70° C., preferably about 60° to about65° C., for about 30 seconds to about 60 minutes, preferably about 10minutes to about 15 minutes. The pasteurized concentrated pulse proteinsolution or resuspended pulse protein precipitate then may be cooled fordrying, preferably to a temperature of about 25° to about 40° C.

Each of the dry pulse protein products obtained by the proceduresdescribed above has a protein content greater than about 60 wt %.Preferably, the dry pulse protein products are isolates with a proteincontent in excess of about 90 wt % protein, preferably at least about100 wt %, (N×6.25) d.b.

The less astringent pulse protein products produced herein are solublein an acidic aqueous environment, making the products ideal forincorporation into beverages, both carbonated and uncarboriated, toprovide protein fortification thereto. Such beverages have a wide rangeof acidic pH values, ranging from about 2.5 to about 5. The pulseprotein products provided herein may be added to such beverages in anyconvenient quantity to provide protein fortification to such beverages,for example, at least about 5 g of the pulse protein per serving. Theadded pulse protein product dissolves in the beverage and the cloud orhaze level of the beverage is not increased by thermal processing. Thepulse protein product may be blended with dried beverage prior toreconstitution of the beverage by dissolution in water. In some cases,modification to the normal formulation of the beverages to tolerate thecomposition of the invention may be necessary where components presentin the beverage may adversely affect the ability of the composition ofthe invention to remain dissolved in the beverage.

EXAMPLES Example 1

This Example illustrates production of the reduced astringency pulseprotein product of the invention utilizing methods where the acidifiedpulse protein solution is partially concentrated or concentrated anddiafiltered prior to the precipitation of the more astringent protein bypH adjustment.

‘a’ kg of ‘b’ was combined with ‘c’ L of reverse osmosis purified (RO)water and the mixture stirred for ‘d’ minutes at ambient temperature.Insoluble material was removed and the sample partially clarified bycentrifugation, yielding a protein solution having a proteinconcentration of ‘e’ wt %. To this protein solution was added ‘f’ kg ofcalcium chloride stock solution, prepared by dissolving 1 kg calciumchloride pellets (95.5%) per 9 L water ‘g’. Insoluble material wasremoved and the sample clarified by centrifugation, yielding ‘h’ L ofprotein extract solution having a protein concentration of ‘i’ wt %. ‘j’L of protein extract solution was combined with ‘k’ L of RO water andthe pH of the sample lowered to ‘l’ with HCl solution (concentrated HCldiluted with an equal volume of water). ‘m’ L of acidified proteinsolution was clarified by running it on a microfiltration systemequipped with a 0.80 μm pore size Membralox ceramic membrane operated at‘n’ ° C. until ‘o’ L of permeate (clarified, acidified protein solution)was collected. ‘p’ L of ‘q’, having a protein content of ‘r’ wt % was‘s’ concentrated to ‘t’ L using a PES ultrafiltration membrane having apore size of 1,000 daltons operated at a temperature of about ‘u’ ° C.‘v’ L of ‘w’ concentrated protein solution was then diafiltered with ‘x’L of RO water at about ‘y’ ° C. to provide ‘z’ of diafiltered, ‘aa’concentrated protein solution having a protein content of ‘ab’ wt %. Thediafiltered, ‘ac’ concentrated protein solution was diluted with ‘ad’ LRO water and the pH adjusted to ‘ae’ with NaOH solution, which causedthe formation of a precipitate. ‘af’ kg of wet precipitate was removedby centrifugation to provide ‘ag’ L of protein solution with a proteincontent of ‘ah’ wt %. The pH of the protein solution was lowered to ‘ai’and then ‘aj’ L of re-acidified protein solution was polished by runningthe solution through a Membralox ceramic microfiltration membrane havinga pore size of 0.80 μm and operated at ‘ak’ ° C. until ‘al’ L ofpermeate was collected. ‘am’ L of ‘an’ was then reduced in volume to‘ao’ L by concentration on a PES ultrafiltration membrane having a poresize of 1,000 daltons operated at a temperature of about ‘ap’ ° C. Theresulting ‘aq’ concentrated protein solution, having a protein contentof ‘ar’ wt % was then diafiltered with ‘as’ L of RO water at about ‘at’° C. ‘au’ to provide ‘av’ kg of concentrated, diafiltered proteinsolution having a protein content of ‘aw’ wt %. This represented a yieldof ‘ax’ % of the protein in the protein extract solution resulting fromthe clarification step after calcium chloride addition. ‘ay’ kg ofconcentrated, diafiltered protein solution was spray dried to yield aprotein product, having a protein content of ‘az’ % (N×6.25) d.b.,termed ‘ba’ ‘bb’.

The ‘af’ kg of wet precipitate collected, having a protein content of‘bc’, represented a yield of ‘bd’ % of the protein in the proteinextract solution resulting from the clarification step after calciumchloride addition. ‘be’ kg of this precipitate was diluted with ‘bf’ kgwater then the pH adjusted to ‘bg’ and the mixture pasteurized at about‘bh’ for ‘bi’ minutes. The ‘bj’ sample was then spray dried to provide adried protein product having a protein content of ‘bk’ % (N×6.25) d.b.that was termed ‘ba’ ‘bl’.

The parameters ‘a’ to ‘bl’ are set forth in the following Table 1.

TABLE 1 Parameters for the production of protein products by theprecipitation fractionation method ba YP20-D23-13A YP20-D24-13AYP20-E02-13A LE03-D02-14A a 30 30 60 36 b yellow pea protein yellow peaprotein yellow pea protein whole green lentil concentrate concentrateconcentrate flour c 500 500 1000 600 d 30 30 30 10 e 2.69 2.68 2.67 1.27f 63.14 65 137.34 80 g and the mixture and the mixture and the mixtureN/A stirred 15 minutes stirred 15 minutes stirred 15 minutes h 459 484978 586 i 1.60 1.41 1.55 0.68 j 459 484 978 586 k 371 317 640 368 l 2.913.12 3.00 3.02 m 830 790 N/A N/A n 59 59 N/A N/A o NR NR N/A N/A p 780700 1585 975 q clarified acidified clarified acidified acidified proteinacidified protein protein solution protein solution solution solution r0.81 0.74 0.81 0.40 s partially N/A N/A partially t 120 72 215 50 u 5757 58 58 v 120 72 215 50 w partially N/A N/A partially x 240 144 430 100y 60 61 59 60 z 120 L 72 L 220 L 48.56 kg aa partially N/A N/A N/A ab4.04 5.57 5.62 5.13 ac partially N/A N/A N/A ad 120 78 344 NR ae 5.635.73 about 5.5 6.10 af 33.50 31.12 105.36 16.14 ag 230.1 128.5 444 80 ah0.40 0.51 0.36 0.65 ai 3.08 2.79 3.11 2.95 aj N/A N/A N/A 80 ak N/A N/AN/A 46 al N/A N/A N/A 64 am 230 150 444 64 an re-acidified proteinre-acidified protein re-acidified protein clarified, re- solutionsolution solution acidified protein solution ao 78 25 32.5 22 ap 58 5254 58 aq N/A N/A N/A partially ar 1.16 1.91 4.62 0.62 as 78 25 32.5 22at 60 59 60 59 au and then further N/A and then further N/A concentratedconcentrated av 34.56 29.14 24.86 21.00 aw 2.87 2.38 6.25 1.51 ax 13.510.1 10.2 8.0 ay 35.54 29.14 24.86 21.00 az 100.17 99.36 101.84 92.26 bbYP705 YP705 YP705 LE705 bc 12.33 11.65 10.38 11.18 bd 56.3 53.2 72.245.2 be 8.5 8.94 24 16.14 bf 8.5 8.94 0 8.00 bg 7.07 6.82 N/A N/A bh N/AN/A N/A 66 bi N/A N/A N/A 15 bj N/A N/A N/A pasteurized bk 102.58 102.49101.44 102.08 bl YP705P YP705P YP705P LE705P NA = not applicable NR =not recorded

Example 2

This Example illustrates production of the reduced astringency pulseprotein product of the invention according to the procedure where theacidified pulse protein solution is pH adjusted to precipitate the moreastringent protein.

18 kg of yellow pea protein concentrate was combined with 300 L ofreverse osmosis purified (RO) water and the mixture stirred for 30minutes at ambient temperature. Insoluble material was removed and thesample partially clarified by centrifugation, yielding a proteinsolution having a protein concentration of 2.47 wt %. To this proteinsolution was added 51.1 kg of calcium chloride stock solution, preparedby dissolving 8.0 kg calcium chloride pellets (95.5%) in 72 L water.Insoluble material as removed and the sample clarified bycentrifugation, yielding 295 L of protein extract solution having aprotein concentration of about 1.32 wt %. The 295 L of protein extractsolution was combined with 206 L of RO water and the pH of the samplelowered to 2.75 with HCl solution (concentrated HCl diluted with anequal volume of water). 495 L of acidified protein solution having aprotein content of 0.66 wt %, was then adjusted to pH 5.5 using 2M NaOHsolution, resulting in the formation of a precipitate. 24.92 kg ofprecipitate was collected by centrifugation yielding 480 L of pulseprotein solution having a protein concentration of 0.20 wt %. The pH ofthe sample was then adjusted to about 3 with diluted HCl solution andthen 480 L of re-acidified pulse protein solution was concentrated to 28L using a PES ultrafiltration membrane having a pore size of 1,000daltons operated at a temperature of about 58° C. 28 L of concentratedprotein solution was then diafiltered with 28 L of RO water at about 63°C. and further concentrated to provide 19.94 kg of concentrated,diafiltered protein solution having a protein content of 6.52 wt %. Thisrepresented a yield of 33.4% of the protein in the protein extractsolution resulting from the clarification step after calcium chlorideaddition. 19.94 kg of concentrated, diafiltered protein solution wasspray dried to yield a protein product, having a protein content of96.07% (N×6.25) d.b., termed YP20-E13-13A YP705.

The 24.92 kg of wet precipitate collected, having a protein content of7.83 wt % represented a yield of 50.1% of the protein in the proteinextract solution resulting from the clarification step after calciumchloride addition. A 14.76 kg aliquot of the precipitate was washed withan equal weight of RO water and then re-captured by centrifugation. Thiswashed precipitate was suspended in fresh water and then spray dried.The dried protein product had a protein content of 95.02% (N×6.25) d.b.and was termed YP20-E13-13A YP705P-01. A second aliquot (10 kg) of theprecipitate was suspended in water and spray dried without a wash step.The dried protein product had a protein content of 87.52 (N×6.25) d.b.and was termed YP20-E13-13A YP705P-02.

Example 3

This Example illustrates production of the reduced astringency pulseprotein product of invention according to the procedure where membraneprocessing is utilized to separate the less astringent proteins from themore astringent proteins. ‘a’ kg of ‘b’ was combined with ‘c’ L ofreverse osmosis purified (RO) water and the mixture stirred for 10minutes at ambient temperature. Insoluble material was removed and thesample partially clarified by centrifugation, yielding a proteinsolution having a protein concentration of ‘d’ wt %. To this proteinsolution was added ‘e’ g antifoam and ‘f’ kg of calcium chloride stocksolution, prepared by dissolving ‘g’ kg calcium chloride pellets (95.5%)in ‘h’ L water. Insoluble material was removed and the sample clarifiedby centrifugation, yielding ‘i’ L of protein extract solution having aprotein concentration of ‘j’ wt %. ‘k’ L of protein extract solution wascombined with ‘l’ L of RO water and the pH of the sample lowered toabout ‘m’ with HCl solution (concentrated HCl diluted with an equalvolume of water). ‘n’ L of acidified pulse protein solution, having aprotein concentration of ‘o’ wt %, was concentrated to ‘p’ using a polyvinylidene fluoride (PVDF) microfiltration membrane having a pore sizeof 0.08 μm operated at a temperature of about ‘q’ ° C. Themicrofiltration retentate was then diafiltered with ‘r’ L of RO water atabout ‘s’ ° C. and then the diafiltered retentate further reduced to ‘t’kg at about ‘u’ ° C. ‘v’ L of microfiltration/diafiltration permeate,having a protein concentration of ‘w’ wt %, was concentrated to ‘x’ Lusing a PES ultrafiltration membrane having a pore size of 1,000 daltonsoperated at a temperature of about ‘y’ ° C. The concentrated proteinsolution was then diafiltered with ‘z’ L of RO water at about ‘aa’ ° C.‘ab’ to provide ‘ac’ kg of concentrated, diafiltered protein solutionhaving a protein content of ‘ad’ wt %. This represented a yield of ‘ae’% of the protein in the protein extract solution resulting from theclarification step after calcium chloride addition. ‘af’ kg ofconcentrated, diafiltered protein solution was spray dried to yield aprotein product, having a protein content of ‘ag’ % (N×6.25) d.b.,termed ‘ah’ ‘ai’.

The ‘aj’ kg of ‘ak’ microfiltration retentate collected, having aprotein content of ‘al’ wt % represented a yield of ‘am’ % of theprotein in the protein extract solution resulting from the clarificationstep after calcium chloride addition. ‘an’ kg of concentrated anddiafiltered microfiltration retentate was adjusted to pH ‘ao’ and thenspray dried to form a protein product having a protein content of ‘ap’ %(N×6.25) d.b., termed ‘ah’ ‘aq’

The parameters ‘a’ to ‘ao’ are set forth in the following Table 2.

TABLE 2 Parameters for the production of protein products by themembrane fractionation method ah YP23-H12-13A YP23-H14-13A YP23-J02-13ALE03-D01-14A a 24 24 60 36 b yellow pea yellow pea yellow pea wholegreen protein protein protein lentil concentrate concentrate concentrateflour c 400 400 1008 600 d 3.11 2.92 3.16 1.25 e N/A N/A 19 N/A f 54.656.0 135 79.36 g 6 6 20 10 h 54 54 180 90 i 398 398.8 934 604 j 1.661.60 about 1.90 0.61 k 398 398.8 934 604 l 269 278.2 666 398 m 3.17 3.162.99 3.01 n 670 490 1440 1025 o 0.86 0.91 0.83 0.30 p 65 L 28.04 kg 180L 35 L q 59 55 55 56 r N/A N/A 180 80 s N/A N/A 55 55 t N/A N/A 140 N/Au N/A N/A 55 N/A v 600 458 about 1470 1052 w 0.18 0.29 0.31 0.27 x 28 3040 48 y 56 54 56 54 z 140 150 200 96 aa 59 59 58 61 ab and further N/Aand further and further concentrated concentrated concentrated ac 21.3632.35 33.6 32.08 ad 3.43 2.44 5.02 2.09 ae 11.0 12.4 9.5 18.2 af 21.3632.35 33.6 32.08 ag 101.64 98.24 99.78 93.52 ai YP706 YP706 YP706 LE706aj 65 L 28.04 kg 140 L 32.12 ak concentrated concentrated concentratedand concentrated diafiltered and diafiltered al 7.02 9.45 6.63 4.87 am69.0 41.5 52.3 42.4 an N/A N/A 135 32.12 ao N/A N/A about 7 7.29 ap N/AN/A 91.60 94.64 aq N/A N/A YP706B LE706B N/A = not applicable

Example 4

This Example contains an evaluation of the dry colour and colour insolution of the reduced astringency pulse protein products produced bythe methods of Examples 1-3.

The colour of the dry powders was assessed using a HunterLab ColorQuestXE instrument in reflectance mode. The colour values are set forth inthe following Table 3:

TABLE 3 HunterLab scores for dry reduced astringency pulse proteinproducts Sample L* a* b* YP20-D23-13A YP705 89.33 0.02 5.75 YP20-D24-13AYP705 88.55 −0.14 5.73 YP20-E02-13A YP705 89.14 0.26 6.68 YP20-E13-13AYP705 86.90 0.90 8.55 LE03-D02-14A LE705 88.09 1.07 5.54 YP23-H12-13AYP706 88.23 −0.09 6.35 YP23-H14-13A YP706 88.53 0.22 6.78 YP23-J02-13AYP706 87.25 0.75 7.45 LE03-D01-14A LE706 85.94 0.84 7.92

As may be seen from Table 3, the reduced astringency pulse proteinproducts were light in colour.

Solutions of the reduced astringency pulse protein products wereprepared by dissolving sufficient protein powder to supply 0.48 g ofprotein in 15 ml of RO water. The pH of the solutions was measured witha pH meter and the colour and clarity assessed using a HunterLab ColorQuest XE instrument operated in transmission mode. The results are shownin the following Table 4.

TABLE 4 pH and HunterLab scores for solutions of reduced astringencypulse protein products sample pH L* a* b* haze YP20-D23-13A YP705 3.3597.2 −0.10 6.42 22.9 YP20-D24-13A YP705 2.93 97.91 −0.40 5.81 8.2YP20-E02-13A YP705 3.39 97.76 −0.33 5.52 9.9 YP20-E13-13A YP705 3.2695.33 0.05 9.69 29.8 LE03-D02-14A LE705 3.21 96.33 0.66 7.18 4.5YP23-H12-13A YP706 3.72 94.65 0.01 9.20 14.9 YP23-H14-13A YP706 3.5796.07 −0.25 8.99 7.7 YP23-J02-13A YP706 3.51 96.55 0.09 9.7 17.2LE03-D01-14A LE706 3.42 93.86 0.60 12.8 21.5

As may be seen from the results in Table 4, the solutions of the reducedastringency pulse protein products were light in colour and, generallylow in haze.

Example 5

This Example contains an evaluation of the solubility in water of thereduced astringency pulse protein products produced by the methods ofExamples 1 and 3. Solubility was tested based on protein solubility(termed protein method, a modified version of the procedure of Morr etal., J. Food Sci. 50:1715-1718) and total product solubility (termedpellet method).

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and wetted by mixing with about 20-25 ml of reverse osmosis (RO)purified water. Additional water was then added to bring the volume toapproximately 45 ml. The contents of the beaker were then slowly stirredfor 60 minutes using a magnetic stirrer. The pH was determinedimmediately after dispersing the protein and was adjusted to theappropriate level (2, 3, 4, 5, 6 or 7) with diluted NaOH or HCl. Asample was also prepared at natural pH. For the pH adjusted samples, thepH was measured and corrected periodically during the 60 minutesstirring. After the 60 minutes of stirring, the samples were made up to50 ml total volume with RO water, yielding a 1% w/v protein dispersion.The protein content of the dispersions was determined by combustionanalysis using a Leco Nitrogen Determinator. Aliquots (20 ml) of thedispersions were then transferred to pre-weighed centrifuge tubes thathad been dried overnight in a 100° C. oven then cooled in a desiccatorand the tubes capped. The samples were centrifuged at 7,800 g for 10minutes, which sedimented insoluble material and yielded a supernatant.The protein content of the supernatant was measured by combustionanalysis and then the supernatant and the tube lids were discarded andthe pellet material dried overnight in an oven set at 100° C. The nextmorning the tubes were transferred to a desiccator and allowed to cool.The weight of dry pellet material was recorded. The dry weight of theinitial protein powder was calculated by multiplying the weight ofpowder used by a factor of ((100−moisture content of the powder(%))/100). Solubility of the product was then calculated two differentways:Solubility (protein method) (%)=(% protein in supernatant/% protein ininitial dispersion)×100  1)Solubility (pellet method) (%)=(1−(weight dry insoluble pelletmaterial/((weight of 20 ml of dispersion/weight of 50 ml ofdispersion)×initial weight dry protein powder)))×100  2)Values calculated as greater than 100% were reported as 100%.

The natural pH values of the 1% w/v protein solutions of the proteinproducts produced in Examples 1 and 3 are shown in Table 5:

TABLE 5 Natural pH of reduced astringency pulse solutions prepared inwater at 1% protein Batch Product Natural pH YP20-D23-13A YP705 3.36YP20-D24-13A YP705 3.15 YP20-E02-13A YP705 3.22 LE03-D02-14A LE705 3.19YP23-H12-13A YP706 3.74 YP23-H14-13A YP706 3.53 LE03-D01-14A LE706 3.40

The solubility results obtained are set forth in the following Tables 6and 7:

TABLE 6 Solubility of products at different pH values based on proteinmethod Solubility (protein method) (%) Batch Product pH 2 pH 3 pH 4 pH 5pH 6 pH 7 Nat. pH YP20- YP705 100 100 95.4 94.4 90.1 96.1 98.1 D23-13AYP20- YP705 98.0 100 100 100 93.7 98.1 100 D24-13A YP20- YP705 96.9 100100 99.0 98.9 93.1 100 E02-13A LE03- LE705 98.0 100 99.1 95.9 100 99.096.1 D02-14A YP23- YP706 99.0 100 100 80.2 78.4 92.9 95.2 H12-13A YP23-YP706 100 100 99.0 73.2 77.8 82.7 100 H14-13A LE03- LE706 93.3 100 10064.6 59.8 64.6 100 D01-14A

TABLE 7 Solubility of products at different pH values based on pelletmethod Solubility (pellet method) (%) Batch Product pH 2 pH 3 pH 4 pH 5pH 6 pH 7 Nat. pH YP20- YP705 97.4 98.8 98.4 94.8 92.9 93.7 98.6 D23-13AYP20- YP705 99.8 100 99.3 98.4 97.4 98.4 99.4 D24-13A YP20- YP705 99.899.8 100 96.4 96.9 97.9 99.1 E02-13A LE03- LE705 99.9 100 99.4 94.4 96.395.7 99.6 D02-14A YP23- YP706 99.8 99.9 99.1 82.0 79.7 87.8 100 H12-13AYP23- YP706 97.8 97.7 98.5 67.9 81.7 75.0 98.9 H14-13A LE03- LE706 96.897.2 96.3 67.1 54.7 68.6 97.4 D01-14A

As can be seen from the results presented in Tables 6 and 7, the reducedastringency pulse protein products were extremely soluble in the pHrange 2-4 and also quite soluble in the pH range of 5-7.

Example 6

This Example contains an evaluation of the clarity in water of thereduced astringency pulse protein products produced by the methods ofExamples 1 and 3.

The clarity of the 1% w/v protein solutions prepared as described inExample 5 was assessed by measuring the absorbance at 600 nm (waterblank), with a lower absorbance score indicating greater clarity.Analysis of the samples on a HunterLab ColorQuest XE instrument intransmission mode also provided a percentage haze reading, anothermeasure of clarity.

The clarity results are set forth in the following Tables 8 and 9:

TABLE 8 Clarity of protein solutions at different pH values as assessedby A600 A600 Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH YP20-YP705 0.008 0.016 0.029 0.337 0.807 0.596 0.022 D23-13A YP20- YP7050.013 0.012 0.021 0.076 0.309 0.213 0.012 D24-13A YP20- YP705 0.0070.011 0.014 0.063 0.506 0.369 0.012 E02-13A LE03- LE705 0.010 0.0120.073 0.062 0.027 0.026 0.014 D02-14A YP23- YP706 0.008 0.016 0.0341.923 1.889 0.791 0.033 H12-13A YP23- YP706 0.011 0.015 0.024 1.9311.690 1.577 0.018 H14-13A LE03- LE706 0.019 0.025 0.050 2.424 2.4122.426 0.024 D01-14A

TABLE 9 Clarity of protein solutions at different pH values as assessedby HunterLab haze analysis HunterLab haze reading (%) Batch Product pH 2pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH YP20- YP705 0.5 5.6 13.0 73.6 90.8 85.39.7 D23-13A YP20- YP705 0.0 1.7 6.4 23.1 65.7 50.3 2.2 D24-13A YP20-YP705 0.0 0.8 3.2 16.0 79.5 68.5 1.0 E02-13A LE03- LE705 0.3 1.2 19.816.7 3.6 1.8 1.8 D02-14A YP23- YP706 0.0 1.0 4.4 96.0 95.8 87.9 4.7H12-13A YP23- YP706 0.0 0.5 2.3 95.9 95.7 95.5 1.1 H14-13A LE03- LE7063.3 4.9 12.6 100.3 101.3 101.3 4.3 D01-14A

As can be seen from the results of Tables 8 and 9, the reducedastringency pulse protein products generally provided transparentsolutions at pH 2-4.

Example 7

This Example contains an evaluation of the solubility in a soft drink(Sprite) and sports drink (Orange Gatorade) of the reduced astringencypulse protein products produced by the methods of Examples 1 and 3. Thesolubility was determined with the protein added to the beverages withno pH correction and again with the pH of the protein fortifiedbeverages adjusted to the level of the original beverages.

When the solubility was assessed with no pH correction, a sufficientamount of protein powder to supply 1 g of protein was weighed into abeaker and wetted by mixing with about 20-25 ml of beverage. Additionalbeverage was then added to bring the volume to 50 ml, and then thesolutions were stirred slowly on a magnetic stirrer for 60 minutes toyield a 2% protein w/v dispersion. The protein content of the sampleswas determined by combustion analysis using a Leco Nitrogen Determinatorthen an aliquot of the protein containing beverages was centrifuged at7,800 g for 10 minutes and the protein content of the supernatantmeasured.Solubility (%)=(% protein in supernatant/% protein in initialdispersion)×100.

Values calculated as greater than 100% were reported as 100%.

When the solubility was assessed with pH correction, the pH of the softdrink (Sprite) and sports drink (Orange Gatorade) without protein wasmeasured. A sufficient amount of protein powder to supply 1 g of proteinwas weighed into a beaker and wetted by mixing with about 20-25 ml ofbeverage. Additional beverage was added to bring the volume toapproximately 45 ml, and then the solutions were stirred slowly on amagnetic stirrer for 60 minutes. The pH of the protein containingbeverages was determined immediately after dispersing the protein andwas adjusted to the original no-protein pH with HCl or NaOH asnecessary. The pH was measured and corrected periodically during the 60minutes stirring. After the 60 minutes of stirring, the total volume ofeach solution was brought to 50 ml with additional beverage, yielding a2% protein w/v dispersion. The protein content of the samples wasdetermined by combustion analysis using a Leco Nitrogen Determinatorthen an aliquot of the protein containing beverages was centrifuged at7,800 g for 10 minutes and the protein content of the supernatantmeasured.Solubility (%)=(% protein in supernatant/% protein in initialdispersion)×100Values calculated as greater than 100% were reported as 100%.

The results obtained are set forth in the following Table 10:

TABLE 10 Solubility of reduced astringency pulse protein products inSprite and Orange Gatorade no pH correction pH correction SolubilitySolubility Solubility (%) in Solubility (%) in (%) Orange (%) OrangeBatch Product in Sprite Gatorade in Sprite Gatorade YP20-D23-13A YP705100 98.0 97.0 100 YP20-D24-13A YP705 100 97.5 99.5 99.0 YP20-E02-13AYP705 100 100 100 100 LE03-D02-14A LE705 100 100 98.5 100 YP23-H12-13AYP706 100 99.0 97.0 96.0 YP23-H14-13A YP706 98.5 99.5 98.0 92.1LE03-D01-14A LE706 92.6 98.9 93.3 100

As can be seen from the results of Table 10, the reduced astringencypulse protein products were highly soluble in the Sprite and the OrangeGatorade.

Example 8

This Example contains an evaluation of the clarity in a soft drink andsports drink of the reduced astringency pulse protein products producedby the methods of Examples 1 and 3.

The clarity of the 2% w/v protein dispersions prepared in soft drink(Sprite) and sports drink (Orange Gatorade) in Example 7 were assessedusing the HunterLab haze method described in Example 6.

The results obtained are set forth in the following Table 11:

TABLE 11 HunterLab haze readings for reduced astringency pulse proteinproducts in Sprite and Orange Gatorade no pH correction pH correctionHaze Haze (%) in Haze Haze (%) in (%) in Orange (%) in Orange BatchProduct Sprite Gatorade Sprite Gatorade no protein 0.0 82.6 0.0 82.6YP20-D23-13A YP705 17.8 70.6 21.8 72.2 YP20-D24-13A YP705 9.4 79.7 12.576.3 YP20-E02-13A YP705 8.5 86.2 20.2 86.5 LE03-D02-14A LE705 1.4 85.41.7 85.0 YP23-H12-13A YP706 10.2 84.7 6.4 79.9 YP23-H14-13A YP706 4.580.6 7.3 78.7 LE03-D01-14A LE706 11.5 77.5 12.1 78.9

As can be seen from the results of Table 11, the addition of the reducedastringency pulse protein products to the soft drink and sports drinkadded little or no haziness.

Example 9

This Example contains an evaluation of the heat stability in water ofthe reduced astringency pulse protein products produced by the methodsof Examples 1 and 3.

2% w/v protein solutions of the protein products were prepared in ROwater. The pH of the solutions was determined with a pH meter and thenadjusted to about 3.0 with HCl solution. The clarity of the solutionswas assessed by haze measurement with the HunterLab Color Quest XEinstrument operated in transmission mode. The solutions were then heatedto 95° C., held at this temperature for 30 seconds and then immediatelycooled to room temperature in an ice bath. The clarity of the heattreated solutions was then measured again.

The clarity of the protein solutions before and after heating is setforth in the following Table 12:

TABLE 12 Effect of heat treatment on clarity of 2% w/v protein solutionsof reduced astringency pulse protein products haze before heat hazeafter heat Batch Product treatment (%) treatment (%) YP20-D23-13A YP70513.0 0.0 YP20-D24-13A YP705 4.2 0.0 YP20-E02-13A YP705 5.5 1.4LE03-D02-14A LE705 1.0 0.0 YP23-H12-13A YP706 5.0 2.0 YP23-H14-13A YP7063.3 2.2 LE03-D01-14A LE706 6.3 1.6

As can be seen from the results in Table 13, the solutions of reducedastringency pulse protein product were substantially clear before heattreatment and the level of haze was actually reduced by the heattreatment.

Example 10

This Example illustrates the production of pulse protein products by themethod described in U.S. patent application Ser. No. 13/556,357.

‘a’ kg of ‘b’ was combined with ‘c’ L of ‘d’ at ‘e’ and agitated for ‘f’minutes. ‘g’ kg of calcium chloride pellets (95.5%) dissolved in ‘h’ Lof RO water was then added and the mixture stirred for an additional ‘i’minutes. The residual solids were removed by centrifugation to produce acentrate having a protein content of ‘j’ % by weight. ‘k’ L of centratewas added to ‘l’ L of RO water at ‘m’ and the pH of the sample loweredto ‘n’ with diluted HCl. The diluted and acidified centrate was furtherclarified by filtration to provide a clear protein solution with aprotein content of ‘o’ % by weight.

The filtered protein solution was reduced in volume from ‘p’ L to ‘q’ Lby concentration on a polyethersulfone membrane, having a molecularweight cutoff of ‘r’ daltons, operated at a temperature of about ‘s’ °C. At this point the protein solution, with a protein content of ‘t’ wt%, was diafiltered with ‘u’ L of RO water, with the diafiltrationoperation conducted at about ‘v’ ° C. The diafiltered protein solutionwas then further concentrated to ‘w’ kg, having a protein content of ‘x’wt %, then diluted with RO water to a protein content of ‘y’ wt % tofacilitate spray drying. The protein solution before spray drying,having a weight of ‘z’ kg was recovered in a yield of ‘aa’ % of theinitial centrate that was further processed. The concentrated anddiafiltered protein solution was then dried to yield a product found tohave a protein content of ‘ab’ wt % (N×6.25) d.b. The product was givendesignation ‘ac’.

The parameters ‘a’ to ‘ac’ are set forth in the following Table 13.

TABLE 13 Parameters for the runs to produce pulse 701 products acYP01-E19-11A YP701 YP05-A18-12A LE01-J24-13A YP701 LE701 a 20 70 20 bYellow split pea flour Yellow split pea whole green lentil flour flour c200 300 200 d 0.15M CaCl₂ RO water 0.13M CaCl₂ e 60° C. 30° C. Ambienttemperature f 30 60 30 g 0 4.52 0 h 0 10 0 i 0 30 0 j 1.32 2.92 1.65 k186.5 223.3 146.2 l 225.8 223.0 147.7 m 60° C. Ambient Ambienttemperature temperature n 3.34 3.04 2.65 o 0.58 1.25 0.62 p 400 550 295q 35 101 25 r 100,000 10,000 100,000 s 58 53 30 t 4.94 4.05 4.23 u 350202 250 v 60 53 32 w 21.52 34.78 21.60 x 7.54 10.02 4.69 y N/A 5.00 N/Az 21.52 57.90 21.60 aa 65.9 44.5 41.9 ab 103.19 101.99 103.11 N/A = notapplicable

Example 11

This Example illustrates a comparison of the astringency level of theYP20-D24-13A YP705 prepared as described in Example 1 with that of theYP01-E19-11A YP701 prepared as described in Example 10.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the YP705 solution was 3.09 and it was adjustedto about 3.50 with food grade sodium hydroxide solution. The initial pHof the YP701 solution was 3.92 and it was adjusted to about 3.50 withfood grade hydrochloric acid. An informal panel of seven panellists wasasked to blindly taste the samples and indicate which was lessastringent.

Five out of seven panellists indicated that the YP20-D24-13A YP705 wasless astringent.

Example 12

This Example illustrates a comparison of the astringency level of theYP20-E02-13A YP705 prepared as described in Example 1 with that of theYP01-E19-11A YP701 prepared as described in Example 10.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the YP705 solution was 3.38 and it was adjustedto about 3.50 with food grade sodium hydroxide solution. The initial pHof the YP701 solution was 3.94 and it was adjusted to about 3.50 withfood grade hydrochloric acid. An informal panel of seven panellists wasasked to blindly taste the samples and indicate which was lessastringent.

Five out of seven panelists indicated that the YP20-E02-13A YP705 wasless astringent.

Example 13

This Example illustrates a comparison of the astringency level of theYP20-E13-13A YP705 prepared as described in Example 2 with that of theYP05-A18-12A YP701 prepared as described in Example 10.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The two samples had pH values within 0.1 units of each other sono pH adjustment was done. An informal panel of eight panelists wasasked to blindly taste the samples and indicate which was lessastringent. The experiment was conducted a second time with a panel often members. The cumulative results are presented below.

Eleven out of eighteen panellists indicated that the YP20-E13-13A YP705was less astringent.

Example 14

This Example illustrates a comparison of the astringency level of theYP20-H12-13A YP706 prepared as described in Example 1 with that of theYP05-A18-12A YP701 prepared as described in Example 10.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the YP706 solution was 3.72 and it was adjustedto about 3.50 with food grade hydrochloric acid. The initial pH of theYP701 solution was 3.17 and it was adjusted to about 3.50 with foodgrade sodium hydroxide solution. An informal panel of seven panellistswas asked to blindly taste the samples and indicate which was lessastringent.

Four out of seven panellists indicated that the YP20-H12-13A YP706 wasless astringent.

Example 15

This Example illustrates a comparison of the astringency level of theYP20-H14-13A YP706 prepared as described in Example 1 with that of theYP05-A18-12A YP701 prepared as described in Example 10.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the YP701 solution was 3.12 and it was adjustedto 3.48 with food grade sodium hydroxide solution. The pH of the YP706solution was 3.46. An informal panel of seven panellists was asked toblindly taste the samples and indicate which was less astringent.

Five out of seven panellists indicated that the YP20-H14-13A YP706 wasless astringent.

Example 16

This Example illustrates a comparison of the astringency level of theLE03-D02-14A LE705 prepared as described in Example 1 with that of theLE01-J24-13A YP701 prepared as described in Example 10.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the LE705 solution was 3.17 and it was adjustedto 3.47 with food grade sodium hydroxide solution. The initial pH of theLE701 solution was 3.81 and it was adjusted to 3.52 with food gradehydrochloric acid. An informal panel of eight panellists was asked toblindly taste the samples and indicate which was less astringent.

Six out of eight panellists indicated that the LE03-D02-14A LE705 wasless astringent.

Example 17

This Example illustrates a comparison of the astringency level of theLE03-D01-14A LE706 prepared as described in Example 3 with that of theLE01-J24-13A LE701 prepared as described in Example 10.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the LE706 solution was 3.37 and it was adjustedto about 3.5 with food grade sodium hydroxide solution. The pH of theLE701 solution was 3.84 and it was adjusted to about 3.5 with food gradehydrochloric acid solution. An informal panel of eight panellists wasasked to blindly taste the samples and indicate which was lessastringent.

Five out of eight panellists indicated that the LE03-D01-14A LE706 wasless astringent.

Example 18

This Example contains an evaluation of the dry colour and colour insolution of the co-products of the production of reduced astringencypulse protein products, prepared according to the methods of Examples1-3.

The colour of the dry powders was assessed using a HunterLab ColorQuestXE instrument in reflectance mode. The colour values are set forth inthe following Table 14:

TABLE 14 HunterLab scores for dry protein products Sample L* a* b*YP20-D23-13A YP705P 84.78 1.30 9.87 YP20-D24-13A YP705P 88.97 0.21 6.08YP20-E02-13A YP705P 89.06 0.22 6.37 YP20-E13-13A YP705P-01 82.64 1.9912.53 YP20-E13-13A YP705P-02 83.61 1.80 11.06 LE03-D02-14A LE705P 74.271.53 8.32 YP23-J02-13A YP706B 81.57 1.32 10.45 LE03-D01-14A LE706B 78.191.96 8.35

As may be seen from the results in Table 14, the co-products generallywere darker, redder and more yellow than the reduced astringency pulseprotein products.

Solutions of the co-products from the preparation of reduced astringencypulse protein products were prepared by dissolving sufficient proteinpowder to supply 0.48 g of protein in 15 ml of RO water. The pH of thesolutions was measured with a pH meter and colour and clarity assessedusing a HunterLab Color Quest XE instrument operated in transmissionmode. The results are shown in the following Table 15.

TABLE 15 pH and HunterLab scores for solutions of pulse protein productssample pH L* a* b* haze YP20-D23-13A YP705P 5.81 43.87 5.5 28.43 97.1YP20-D24-13A YP705P 6.13 40.94 6.82 30.44 97.3 YP20-E02-13A YP705P 4.9539.68 6.79 31.08 99.2 YP20-E13-13A YP705P-01 5.29 39.32 8.4 33.01 96.5YP20-E13-13A YP705P-02 5.03 32.10 10.7 34.12 96.4 LE03-D02-14A LE705P6.40 11.69 11.81 17.59 97.9 YP23-J02-13A YP706B 7.39 41.26 7.88 31.6595.7 LE03-D01-14A LE706B 7.09 38.09 7.75 25.18 97.3

As may be seen from the results in Table 15, the solutions of theco-products were all very high in haze. The solutions were also darker,redder and more yellow than the solutions of the reduced astringencypulse products.

Example 19

This Example contains an evaluation of the solubility in water of theco-products of the production of the reduced astringency pulse products,prepared by the methods of Examples 1 and 3. Solubility was tested basedon protein solubility (termed protein method, a modified version of theprocedure of Morr et al., J. Food Sci. 50:1715-1718).

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and wetted by mixing with about 20-25 ml of reverse osmosis (RO)purified water. Additional water was then added to bring the volume toapproximately 45 ml. The contents of the beaker were then slowly stirredfor 60 minutes using a magnetic stirrer. The pH was determinedimmediately after dispersing the protein and was adjusted to theappropriate level (6, 6.5, 7, 7.5 or 8) with diluted NaOH or HCl. The pHwas then measured and corrected periodically during the 60 minutesstirring. After the 60 minutes of stirring, the samples were made up to50 ml total volume with RO water, yielding a 1% w/v protein dispersion.The protein content of the dispersions was determined by combustionanalysis using a Leco Nitrogen Determinator. The samples were thencentrifuged at 7,800 g for 10 minutes, which sedimented insolublematerial and yielded a supernatant. The protein content of thesupernatant was measured by combustion analysis.

Solubility of the product was then calculated:Solubility (protein method) (%)=(% protein in supernatant/% protein ininitial dispersion)×100  1)Values calculated as greater than 100% were reported as 100%.

The solubility results obtained are set forth in the following Table 16:

TABLE 16 Solubility of products at different pH values based on proteinmethod Solubility (protein method) (%) Batch Product pH 6 pH 6.5 pH 7 pH7.5 pH 8 YP20-D23-13A YP705P 5.7 2.9 9.9 12.0 11.8 YP20-D24-13A YP705P13.0 9.9 15.2 11.7 15.3 LE03-D02-14A LE705P 13.6 10.9 11.0 11.7 9.6YP23-J02-13A YP706B 16.5 15.5 20.4 17.7 19.6 LE03-D01-14A LE706B 2.0 1.84.7 9.3 5.1

As may be seen from the results in Table 16, the co-products of theproduction of the reduced astringency pulse protein products were poorlysoluble over the pH range of 2 to 7.

Example 20

This Example contains an evaluation of the water binding capacity of theco-products of the production of the reduced astringency pulse products,prepared by the methods of Examples 1 and 3.

Protein powder (1 g) was weighed into centrifuge tubes (50 ml) of knownweight. To this powder was added approximately 20 ml of reverse osmosispurified (RO) water at the natural pH. The contents of the tubes weremixed using a vortex mixer at moderate speed for 1 minute. The sampleswere incubated at room temperature for 5 minutes then mixed with thevortex mixer for 30 seconds. This was followed by incubation at roomtemperature for another 5 minutes followed by another 30 seconds ofvortex mixing. The samples were then centrifuged at 1,000 g for 15minutes at 20° C. After centrifugation, the supernatant was carefullypoured off, ensuring that all solid material remained in the tube. Thecentrifuge tube was then re-weighed and the weight of water saturatedsample was determined.

Water binding capacity (WBC) was calculated as:WBC (ml/g)=(mass of water saturated sample−mass of initial sample)/(massof initial sample×total solids content of sample)

The water binding capacity results obtained are set forth in thefollowing Table 17.

TABLE 17 Water binding capacity of various products product WBC (ml/g)YP20-D23-13A YP705P 2.60 YP20-D24-13A YP705P 2.59 LE03-D02-14A LE705P3.90 YP23-J02-13A YP706B 2.88 LE03-D01-14A LE706B 2.74

As may be seen from the results of Table 17, all of the co-products ofthe production of the reduced astringency pulse protein products hadmoderate water binding capacities.

Example 21

This Example illustrates the preparation of a pulse protein isolate byconventional isoelectric precipitation.

20 kg of yellow pea protein concentrate was added to 200 L of RO waterat ambient temperature and the pH adjusted to about 8.5 by the additionof sodium hydroxide solution. The sample was agitated for 30 minutes toprovide an aqueous protein solution. The pH of the extraction wasmonitored and maintained at about 8.5 throughout the 30 minutes. Theresidual pea protein concentrate was removed and the resulting proteinsolution clarified by centrifugation and filtration to produce 240 L offiltered protein solution having a protein content of 3.52% by weight.The pH of the protein solution was adjusted to about 4.5 by the additionof HCl that had been diluted with an equal volume of water and aprecipitate formed. The precipitate was collected by centrifugation thenwashed by re-suspending it in 2 volumes of RO water. The washedprecipitate was then collected by centrifugation. A total of 30.68 kg ofwashed precipitate was obtained with a protein content of 22.55 wt %.This represented a yield of 81.9% of the protein in the clarifiedextract solution. An aliquot of 15.34 kg of the washed precipitate wascombined with 15.4 kg of RO Water and then the pH of the sample adjustedto about 7 with sodium hydroxide solution. The pH adjusted sample wasthen spray dried to yield an isolate with a protein content of 90.22%(N×6.25) d.b. The product was designated YP12-K13-12A conventional IEPpH 7.

Example 22

This Example is a sensory evaluation of the YP20-D23-13A YP705P productprepared as described in Example 1 with the conventional pea proteinisolate product prepared as described in Example 21.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the YP12-K13-12A conventional IEP pH 7 solutionwas 7.08. The initial pH of the YP705P solution was 5.77 and it wasadjusted to 7.08 with food grade sodium hydroxide solution. An informalpanel of eight panellists was asked to blindly taste the samples andindicate which had a cleaner flavour and which sample they preferred.

Seven out of eight panellists preferred the YP20-D23-13A YP705P andseven out of eight found it to have a cleaner flavour.

Example 23

This Example is a sensory evaluation of the YP20-D24-13A YP705P productprepared as described in Example 1 with the conventional pea proteinisolate product prepared as described in Example 21.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The initial pH of the YP12-K13-12A conventional IEP pH 7 solutionwas 7.06. The initial pH of the YP705P solution was 6.18 and it wasadjusted to 7.10 with food grade sodium hydroxide solution. An informalpanel of nine panellists was asked to blindly taste the samples andindicate which had a cleaner flavour and which sample they preferred.

All nine panellists preferred the YP20-D24-13A YP705P and found it tohave a cleaner flavour.

Example 24

This Example is a sensory evaluation of the YP23-J02-13A YP706B productprepared as described in Example 3 with the conventional pea proteinisolate product prepared as described in Example 21.

Samples were prepared for sensory evaluation by dissolving sufficientprotein powder to supply 5 g protein in 250 ml of purified drinkingwater. The pH of the YP12-K13-12A IEP pH 7 solution was 7.09. The pH ofthe solution of YP23-J02-13A YP706B was adjusted to 7.04 with food gradehydrochloric acid. An informal panel of eight panellists was asked toblindly taste the samples and indicate which had a cleaner flavour andwhich sample they preferred. The experiment was conducted a second timewith a panel having 7 members. The cumulative results are presentedbelow.

Eleven out of fifteen panellists found the YP23-J02-13A YP706B to havethe cleaner flavour. Ten out of fifteen panellists preferred theYP23-J02-13A YP706B.

Example 25

This Example illustrates the molecular weight profile of the pulseprotein products prepared as described in Examples 1-3 as well as themolecular weight profile of some commercial yellow pea protein products(Propulse (Nutri-Pea, Portage la Prairie, MB), Nutralys S85F (RoquetteAmerica, Inc. Keokuk, Iowa) and Pisane C9 (Cosucra Groupe Warcoing S.A.,Belgium). These protein products were chosen as they are among the mosthighly purified pea protein ingredients currently commerciallyavailable.

Molecular weight profiles were determined by size exclusionchromatography using a Varian ProStar HPLC system equipped with a300×7.8 mm Phenomenex BioSep S-2000 series column. The column containedhydrophilic bonded silica rigid support media, 5 micron diameter, with145 Angstrom pore size.

Before the pulse protein samples were analyzed, a standard curve wasprepared using a Biorad protein standard (Biorad product #151-1901)containing proteins with known molecular weights between 17,000 Daltons(myoglobulin) and 670,000 Daltons (thyroglobulin) with Vitamin B12 addedas a low molecular weight marker at 1,350 Daltons. A 0.9% w/v solutionof the protein standard was prepared in water, filtered with a 0.45 μmpore size filter disc then a 50 μL aliquot run on the column using amobile phase of 0.05M phosphate/0.15M NaCl, pH 6 containing 0.02% sodiumazide. The mobile phase flow rate was 1 mL/min and components weredetected based on absorbance at 280 nm. Based on the retention times ofthese molecules of known molecular weight, a regression formula wasdeveloped relating the natural log of the molecular weight to theretention time in minutes.Retention time (min)=−0.955×1n (molecular weight)+18.502(r ²=0.98)

For the analysis of the pulse protein samples, 0.05M NaCl, pH 3.5containing 0.02% sodium azide was used as the mobile phase and also todissolve dry samples. Protein samples were mixed with mobile phasesolution to a concentration of 1% w/v, placed on a shaker for at least 1hour then filtered using 0.45 μm pore size filter discs. Sampleinjection size was 50 μL. The mobile phase flow rate was 1 mL/minute andcomponents were detected based on absorbance at 280 nm.

The above regression formula relating molecular weight and retentiontime was used to calculate retention times that corresponded tomolecular weights of 100,000 Da, 15,000 Da, 5,000 Da and 1,000 Da. TheHPLC ProStar system was used to calculate the peak areas lying withinthese retention time ranges and the percentage of protein ((range peakarea/total protein peak area)×100) falling in a given molecular weightrange was calculated. Note that the data was not corrected by proteinresponse factor.

The molecular weight profiles of the products prepared as described inExamples 1-3 and the commercial products are shown in Table 18.

TABLE 18 Molecular weight profile of pulse protein products product% >100,000 Da % 15,000-100,000 Da % 5,000-15,000 Da % 1,000-5,000 DaYP20-D23-13A YP705 31 33 31 5 YP20-D24-13A YP705 30 36 29 5 YP20-E02-13AYP705 31 37 28 4 YP20-E13-13A YP705 66 16 14 4 LE03-D02-14A LE705 37 3816 9 YP23-H12-13A YP706 21 30 42 7 YP23-H14-13A YP706 28 29 36 7YP23-J02-13A YP706 16 28 48 8 LE03-D01-14A LE706 39 34 18 9 YP20-D23-13AYP705P 22 29 34 15 YP20-D24-13A YP705P 21 30 33 17 YP20-E02-13A YP705P24 32 30 15 YP20-E13-13A YP705P-01 27 26 19 29 YP20-E13-13A YP705P-02 3822 17 24 LE03-D02-14A LE705P 35 37 22 6 YP23-J02-13A YP706B 38 28 14 20LE03-D01-14A LE706B 75 16 3 5 Nutralys S85F 7 29 9 56 Pisane C9 5 31 2936 Propulse 13 25 18 45

As may be seen from the results presented in Table 18, the molecularweight profiles of the products prepared according to Examples 1-3 weredifferent from the molecular weight profiles of the commercial yellowpea protein products.

Example 26

This Example contains an evaluation of the phytic acid content of thepulse protein products produced as described in Examples 1 to 3. Phyticacid content was determined using the method of Latta and Eskin (J.Agric. Food Chem., 28: 1313-1315).

The results obtained are set forth in the following Table 19.

TABLE 19 Phytic acid content of protein products product % phytic acidd.b. YP20-D23-13A YP705 0.00 YP20-D24-13A YP705 0.00 YP20-E02-13A YP7050.02 YP20-E13-13A YP705 0.00 LE03-D02-14A LE705 0.19 YP23-H12-13A YP7060.00 YP23-H14-13A YP706 0.00 YP23-J02-13A YP706 0.01 LE03-D01-14A LE7060.29 YP20-D23-13A YP705P 0.02 YP20-D24-13A YP705P 0.01 YP20-E02-13AYP705P 0.06 YP20-E13-13A YP705P-01 0.00 YP20-E13-13A YP705P-02 0.00LE03-D02-14A LE705P 0.23 YP23-J02-13A YP706B 0.10 LE03-D01-14A LE706B0.21

As may be seen from the results in Table 19, all of the products testedwere low in phytic acid content.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides pulseprotein products, preferably pulse protein isolates, which have reducedastringency when tasted in an acidic solution such as an acidicbeverage. Modifications are possible within the scope of this invention.

What we claim is:
 1. A pulse protein product which is pea or lentil andwhich has a protein content of at least about 60 wt % (N×6.25) d.b.which has a solubility at 1% protein w/v in water at a pH of about 2 toabout 7 of greater than about 50%, as determined by the protein methodor the pellet method as calculated from the formulae:Solubility (protein method) (%)=(% protein in supernatant/% protein ininitial dispersion)×100  1)Solubility (pellet method) (%)=(1−(weight dry insoluble pelletmaterial/((weight of 20 ml of dispersion/weight of 50 ml ofdispersion)×initial weight dry protein powder)))×100.  2)
 2. The pulseprotein product of claim 1 which has a protein content of at least about90 wt % (N×6.25) d.b.
 3. The pulse protein product of claim 1 which hasa protein content of at least about 100 wt % (N×6.25) d.b.